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REVIEW OF LITERATURE

Breast cancer is by far the most common cancer diagnosed in women in the world. Breast

cancer ranks second and cervical cancer ranks seventh according to incidence of cancers in

the world11. Cancers of uterine cervix and breast are the two leading cancers sites among

Indian women with 13,4420 incident cases, 338,010 five yearly prevalence and 115,251

incident cases, 315,679 five yearly prevalence respectively. According to National Cancer

Registry Program (NCRP) recent report for the 2008, the load of breast and cervical cancers

together was 23.6-38.7% of total cancers in North Eastern states while in all the other states

these two cancers contributed 35.2-57.7% of the total cancers. Published reports from

different cancer registries in India indicate rising trends in breast cancer incidence.12

The current data support that various factors like marital status, place of residence

(urban/rural), BMI, and breast-feeding were significantly associated with breast cancer. The

incidence of breast cancer is on the rise in India. It is the second most common cancer in

India and accounts for 7% of global burden of breast cancer and 20% of all cancers in women

of India. It is estimated that in 2001 there were approximately 80,000 new breast cancer cases

in India12. Breast cancer is the most common cancer in women in Delhi and accounts to about

26.8% of all cancers in women in Delhi15

Geographic variation in breast cancer incidence can be attributed to racial and genetic

differences, cultural differences as well as environmental exposures that are variable

throughout the world.

Locally advanced breast cancer constitutes more than 50% to 70% of patients presenting for

treatment.17 It has been found in a study that 84% patients belonged to stage III/IV at

presentation.17 The findings reveal that the breast carcinoma frequently presents at a higher

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stage in India. This is in contrast to that reported from developed countries where 58 % were

stage I while only 33% were stage II disease.

MOLECULAR GENETICS OF BREAST CANCER

In breast tissue, the hormone-sensitive cells in the terminal duct-lobular unit contain the stem

cells that generate the lactating lobules. These cells are responsive to estrogen and

progesterone, which provide signals for growth during the menstrual cycle and elicit

proliferation during pregnancy. Should the individual carry a germline or somatic mutation in

tumor supressor genes, the stem cells in the terminal ductal-lobular unit are predisposed to

malignancy, but these cells are quiescent in prepubertal life, and no tumor can form. When

these cells are subjected to hormone stimulation during puberty, their DNA is replicated to

permit cell proliferation. However, if there is a genetic defect in p53 or in the other genes, the

control and regulation of replication cannot be carried out in a proper manner. Therefore,

cells start proliferating in an uncontrolled way, thus causing instability and activation of

proto-oncogenes.

Some oncogenes also initiate gene amplifications (erbB2, c-myc, int-2), leading to

tumorogenesis.14 Although the activation of oncogenes has clear relevance in selected breast

cancer cases, a more common finding in breast cancer cells is a mutation in one or more

tumor suppressor genes. As a class, these genes function to maintain genomic integrity and

help prevent the propagation of damaged DNA. Aberration in many tumor suppressor genes

directly affects cellular susceptibility to DNA damage and cellular capacity DNA damage

repair. Others recognize damaged DNA and promote cell cycle arrest, allowing for repair of

damage before DNA synthesis and mitosis commence. Finally, tumor suppressor gene

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products may also inhibit propagation of damaged DNA by inducing apoptotic cellular

death.18

Although it has been demonstrated that mutations of p53, BRCA1 and BRCA2 can lead to

increased breast cancer risk, the percentage of these mutations is comparatively low in breast

cancer cases (only 6% to 8% of the total United States breast cancer population). In addition,

these genes are not found in all cases of familial breast cancer.

CARCINOGENESIS:

Breast cancer is believed to arise from series of sequential mutations that occur as a result of

genomic instability and or environmental factors 19. There is a sequential progression through

clinical and pathological stages starting with carcinoma in situ, progressing into invasive

carcinoma and then finally into metastasis20. According to the multistep hypothesis,

progression in the tumor stage is associated with the sequential acquisition of various genetic

and consequently phenotypic alterations in a single cell followed by clonal selection and

expansion.

The successive clonal populations are thought to acquire an increasing aggressiveness, with

alterations in various properties such as proliferation, adhesion, proteolysis, motility,

angiogenic ability etc. and loss of estrogen receptor20. Considerable intratumor diversity may

result from the co-existence of these clonal populations.

MICROSCOPIC ANATOMY OF ADULT BREAST:

The mammary gland begins to develop in early puberty when the primitive ductal structures

enlarge and branch. The main functional units of the human breast are terminal duct lobular

units, which contains two types of cells: luminal (glandular) and basal21. The majority are

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columnar or cuboidal cells lining the lumen. They have cytoplasm endowed with abundant

organelles involved in secretion. Myoepithelial cells lie between the epithelial layer and the

basal lamina, where they form a network of slender processes investing the overlying

epithelial cells.

The branching network of myoepithelial cell cytoplasmic processes can be seen especially

well in scanning electron micrographs of lobules taken after the basal lamina and surrounding

collagen have been removed by enzyme digestion22. Spindle-shaped ductal myoepithelial

cells lie parallel to the long axis of the duct and form a continuous layer. Contraction of

myoepithelial cells in lobules and around ducts contributes to the flow of milk during

lactation23.

The histologic appearance and immunoreactivity of myoepithelial cells are variable,

especially in pathologic conditions, and depend on the degree to which the myoid or

epithelial phenotype is accentuated in a particular situation. Myoepithelial cells usually

display nuclear reactivity for P63, which is the most useful marker for detecting these cells in

normal and lesional tissue. Epithelioid myoepithelial cells can have reduced P63 reactivity.

Other myoepithelial markers include actin, CK5/6, CD10, and calponin.

The epithelial–stromal junction consists of the epithelial–myoepithelial layer within the duct,

the basal lamina, and a surrounding zone of delimiting fibroblasts and capillaries. Elastic

tissue fibers are variably present around normal ducts and these fibers tend to be less

pronounced in the pre-menopausal breast. Farahmand and Cowan23 were able to detect

periductal elastic fibers in 71% of patients younger than 50 years of age and in 89% of

patients older than 50 years. Marked periductal elastic fiber deposition was found in only 3%

of normal specimens from women younger than 50 years and in 17% of woman older than

age 50. Elastic fibers are largely absent at the lobular level, and, when present, they surround

but do not extend into the lobular unit.

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In addition to elastic fibers, the normal periductal stroma contains a sparse scattering of

lymphocytes, plasma cells, mast cells, and histiocytes.

CLASSIFICATION OF BREAST CARCINOMAS:

The classification of breast carcinomas is fundamental to understanding and treatment of the

disease, with the prognosis of the disease being directly related to stage, grade and subtype.

Although there are many different types of breast cancer, most tumors (75-80%) are

categorized in single histological category-invasive ductal carcinoma not otherwise specified

(NOS) 24. This category is a heterogeneous group of cancers that do not show significant

common characteristics allowing them to be placed in one of the specialized categories.

Morphological classification:

1) Invasive ductal carcinoma NOS: This is the largest group constituting 65-80% of

mammary carcinomas25.The WHO definition of this category is that of exclusion 26. It

includes tumors that express in part one or more features of the specific types of

breast carcinoma but do not constitute pure examples of the individual tumors.

Tumors combining IDC with pagets disease is also included in this category.

2) Tubular carcinoma: defined as a highly differentiated invasive carcinoma with cells

arranged in well defined tubules typically one layer thick and surrounded by abundant

fibrous stroma and this accounting for atleast 75% of the growth 26. It constitutes less
25,27
than 2% of all breast carcinomas and tend to be smaller in size. Patients tend to

be younger with median age of mid to late forties 27. It has a favourable prognosis with

the average frequency of lymph node metastasis being 10%.

3) Medullary carcinoma: WHO defines it as a well circumscribed carcinoma composed

of poorly differentiated cells with scant stroma and prominent lymphoid infiltration 26.

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The various characteristics include: lymphoplasmacytic reaction, microscopic

circumscription, syncytial growth pattern, poorly differentiated nuclear grade and

high mitotic rate28,29 .The characteristic immunophenotype reported in various studies,

is P53 positivity along with HER2 negativity30 and less than 10% of these are estrogen

and progesterone receptor positive31. Patients tend to have overall lower frequency of

axillary node metastasis and better prognosis than IDC patients31

4) Metaplastic carcinoma: Carcinomas with metaplasia usually lack prominent

glandular epithelial cells, therefore they have low levels of estrogen receptor 32. The

immunohistochemical results are consistent with epithelial or myoepithelial origin 33.

There is coexpression of cytokeratin and vimentin in spindle cell elements of

metaplastic carcinomas34, however cytokeratin expression is diminished in vimentin

positive cell lines35. Vimentin expression is absent from non tumorigenic human

mammary epithelial cells but present in highly tumorigenic cell line. These altered

cells are characterized by diminished keratin 19 expression and diminished cell

adhesion35.The majority of metaplastic carcinomas are hormone receptor negative34.

5) Mucinous carcinoma: constitute not more than 2% of all tumors 36. It is reported to

occur in older patients, constituting about 7% of carcinomas in women 75 years or

older and only 1% among those younger than 35 years 36. About 60% are estrogen

receptor positive when studied biochemically and immunohistochemical studies have

shown receptor activity in nearly 90% of cases37. Intraductal carcinoma is associated

in about 75% of the lesions. They contain acidic and neutral mucopolysaccharides and

have relatively favourable prognosis. 36-39

6) Secretory carcinoma: The median age of patients of this rare type is 25 years. On IHC,

the estrogen receptors expression is negative in 14 tumors while 5 tumors were

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positive40, seven carcinomas were PR positive and negative in 3 tumors 41,42. The tumor

cells, glands and microcystic spaces contain abundant secretion reacting positively for

mucin and PAS. Strong positive staining of cells but not secretion is seen for alpha-

lactalbumin as well as S-100 protein 42 and CEA. In majority of the patients, it is a low

grade variant with exceptionally favourable prognosis.

7) Lobular carcinoma: The frequency of invasive lobular carcinoma has been reported as

high as 10-14% 42with the mean age of patient being 57 years. Patients tend to have a

higher frequency of bilateral carcinoma. Lymphatic tumor emboli and blood vessel

invasion is rarely noted43, but perineural invasion is not uncommon. IHC shows

estrogen receptor positivity in all cases of invasive lobular carcinoma including

classical and variant types44. In addition, a substantial number of these tumors are

reactive for CEA. While Her2/neu is rarely detected in in-situ or invasive lobular

carcinoma43. The 10 year survival rate of stage I patients is comparable (72%) to that

of stage I of IDC patients, while it is worse (only 9.3%) in stage II patients than with

high grade IDC of similar stage44.

Biological characteristics differ between the histological types. Papillary and mucinous

carcinomas tend to occur in older patients compared to those with other types of

carcinoma. On the other hand, medullary carcinomas occur in the relatively young, and

they have poorly differentiated histology, lymphocytic infiltration and absence of

hormone receptors. Lobular cancer tends to have contra-lateral recurrence .Histological

types associated with favourable prognosis include tubular (>97% 5- year disease free

survival), mucinous (84%), medullary (78%) carcinomas.

HISTOPATHOLOGICAL PROGNOSTIC FACTORS:

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Breast carcinoma is a heterogeneous disease clinically and pathologically. The outcome for

women with breast cancer varies widely.

A multitude of parameters are used to predict the clinical outcome of breast carcinoma and to

guide the treatment accordingly. The most important prognostic factors in current use are

features such as lymph node status, tumor size and tumor grade. Prognostic information is

important in counselling the patients about the likely outcome of their disease and in selecting

appropriate treatment45.

Modified Bloom Richardson grading46: The parameters measured are the extent of tubule

formation, nuclear pleomorphism and mitotic rate. The most important feature of this grading

is the mitotic rate47. When tumors of equivalent stage are compared, high grade tumors (less

differentiated, lacking tubule formation, marked nuclear pleomorphism and high rate of

mitosis) are linked with worse prognosis when compared with low grade tumors (well

differentiated, showing widespread tubule formation and minimal nuclear pleomorphism and

a lower mitotic count) 48. Studies have demonstrated that patients with high grade or poorly

differentiated invasive duct carcinoma have a significantly higher frequency of axillary

lymph node metastasis and recurrences49.

Tumor necrosis: The independent prognostic significance of tumor necrosis has been

commented upon in various studies. The conclusions are somewhat conflicting but most have

indicated that the presence of tumor necrosis is a poor prognostic feature and associated with

reduced overall survival1, 51.

Stromal lymphoplasmacytic infiltration: The prognostic significance of stromal

lymphoplasmacytic infiltration within and around the invasive duct carcinomas has been the

subject of considerable interest. The reaction consists of mature lymphocytes predominantly

T lymphocytes with a variable admixture of plasma cells. Most non medullary duct

carcinomas with a prominent lymphocytic reaction tend to be poorly differentiated and have a

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circumscribed rather than infiltrative contour49.Medullary carcinomas and invasive duct

carcinoma with a marked lymphocytic reaction are almost always estrogen and progesterone

receptor negative49.While the favourable prognosis of medullary carcinoma has been ascribed

to the lymphoplasmacytic reaction, it is less clear that the same conclusion can be drawn

about non-medullary invasive duct carcinomas49.

Lymphatic tumor emboli: The presence of lymphatic tumor emboli in the breast carcinoma is

an unfavourable prognostic finding. Extratumoral lymphatic tumor emboli in the breast are

found associated with approximately 15% of invasive duct carcinomas. The majority of these

patients also have axillary lymph node metastasis49.

Blood vessel invasion: The reported frequency of blood vessel invasion varies from 4% to

47.2% in invasive breast carcinomas49, 52, 53. Vascular invasion has been shown in a number of

studies, to be a powerful predictor of local recurrence following conservation therapy54.

Adipose tissue invasion(ATI): ATI is one of the biologic indicators of tumor aggressiveness.

It has been seen that ATI is an independent predictor of nodal metastasis. The patients with

ATI have poor prognosis as compared to patients without ATI10.

Angiogenic capacity: The angiogenic capacity of breast carcinomas has attracted interest as a

prognostic indicator. The capacity of angiogenesis is attributed to the elaboration of VEGF

and vascular permeability factor (VPF) and basic fibroblast growth receptor49 (bFGF).

Stromal factors: It is not clear whether the character of stroma in an invasive duct carcinoma

is an independent prognostic variable. Tumors that contain minimal stromal reaction tend to

have the following characteristics: circumscription poorly differentiated nuclear and

histological grade and a prominent lymphoplasmacytic infiltrate 49. They also tend to be

estrogen receptor negative. On the other hand, densely fibrotic or scirrhous carcinomas are

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more likely to be stellate and moderately differentiated with little lymphoplasmacytic

reaction. A greater of these lesions is estrogen receptor positive49.

Stromal elastosis: It is a feature of many benign and malignant breast lesions. The frequency

of the most extreme degrees of elastosis is described in 17% to 23 % of breast

carcinomas56.57.There is a significant correlation between the presence of marked elastosis and

both estrogen receptor expression and response of patients to endocrine therapy. This is

probably due to the fact that elastosis is particularly associated with tumor types having a

relatively good prognosis such as tubular and invasive cribriform carcinoma58.

Although these histological prognosticators are undeniably useful, the clinical course of any

individual patient with breast carcinoma remains difficult to predict.

MOLECULAR CLASSIFICATION OF BREAST CARCINOMA

Recently, hormone receptors and variable cytokeratin expression has led to the classification

of breast cancers into distinct subgroups: Luminal, normal breast like, Her2/neu positive and

basal like subtypes, also based on global mRNA expression profiles48.

Luminal A: (40-55% of cancers): This is the largest group and consists of cancers that are

ER positive, Her2/neu negative and express one or more of the luminal cytokeratins (CK

8/18,CK19). ER positive carcinomas show increased transcription of genes thought to be

characteristic of normal luminal cells. The majority are well or moderately differentiated and

most occur in postmenopausal women45.

These cancers are generally slow growing and respond well to hormonal treatments.

Conversely, only a small number will respond to standard chemotherapy45.

Luminal B: (15-20% of cancers): This group of cancers also express ER and CK8/18 and/or

CK19 but is generally of higher grade, has a higher proliferative rate and often overexpresses

Her2/neu. They are sometimes referred to as triple positive cancers 45. They compose a major

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group of ER positive cancers that are more likely to have lymph node metastasis and that may

respond to chemotherapy.

Her2neu positive: (7-12% of carcinomas): This group comprises ER and CK negative

carcinomas that overexpress Her2/neu protein. In over 90% of Her2/neu positive cancers,

overexpression is due to amplification of the segment of DNA on 17q21 that includes the

Her2neu gene. In rare cases, Her2neu protein overexpression may occur as a result of

mechanism other than gene amplification. These cancers are poorly differentiated, have a

high proliferation rate and are associated with a high frequency of brain metastasis45.

Normal breast like: (6-10% of cancers): This is a small group of usually well differentiated

ER positive and Her2neu negative cancers characterized by the similarity of their gene

expression pattern to normal tissue. There is expression of both luminal (CK8/18, CK19) and

basal cytokeratins (CK 5/6, CK14). It is not yet clear whether or not this is a specific tumor

expression45.

Basal like breast carcinoma: comprises of 15-25% of breast cancers, and it has been

correlated with aggressive behaviour, poor prognosis and better response to chemotherapy

than conventional breast carcinoma45. The tumors often affect younger patients, frequently

lack expression of hormone receptors ER, PR and Her2neu, and express markers typical of

myoepithelial cells (basal keratins, P-cadherin, p63 or laminin), progenitor cells or putative

stem cells (CK5/6)124.

Importantly, these various breast cancer subtypes are associated with markedly different

clinical outcomes, ranging from the best prognosis luminal A tumors to the worst prognosis

Her2 and basal-like tumors91.

HORMONAL DIFFERENTIATION:

The Immunohistochemical demonstration of hormone receptors has become a routine

practice in every patient diagnosed with breast cancer. This may be attributed to the

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convenience, cost effectiveness and its easy applicability to paraffin processed material59.

Steroid receptors in breast:

The ovarian steroids estrogen and progesterone are essential for mammary growth and

development, however the mechanisms by which they influence the proliferative activity of

the mammary epithelium remains unclear.

The level of receptors is higher when there is high epithelial cellularity and also in more

recent lesions. The level and subcellular location of the receptors also depends on the

endogenous secretion of steroids: their level varying throughout the menstrual cycle, being

maximal at the end of the follicular phase, due to the high estradiol secretion 60. The cytosol

and nuclear level and location of the receptors are also modified by the hormonal therapy in

the form of estrogen-progestagens given as a contraceptive, or progestins given as a

substitutive and antiestrogen therapy60.

The expression level and staining patterns of several proteins are also useful in predicting

which tumors will respond to specific therapies; Tamoxifen is used to treat only estrogen

receptor positive tumors and herceptin to Her2/neu overexpressing tumors.

Both ER and PR are highly associated with age at diagnosis, being positive more often in

tumors from postmenopausal than in premenopausal women. The proportional increase in ER

and PR positivity with age is more marked for PR than ER61. The expression of ER and PR

has not been related to major risk factors for developing breast carcinoma such as family

history of breast carcinoma, parity and age of menarche62.

The proportion of both ER/PR positive cases is low in Indian studies. Dutt 63 et al found both

ER/PR expression in only 33% cases out of which 24% were ER positive and 30% expressed

PR. It further demonstrated that ER/PR expression increases with advancing age. Similar

findings were obtained by Desai64 et al who found that only 25% cases out of 798 breast

cancers expressed both ER and PR. Individually PR expression was seen in 46.1% of cases.

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The recent study by Tanuja65 et al documented a total hormone receptor expression of only

53.5 % in breast cancers in Indian patients as opposed to 75-80% reported in the western

countries66.

On correlation of ER/PR status with nodal metastasis, it has been observed that metastatic

tumors are more often hormone receptor negative than primary tumors. Values range from

30.8-58% in node negative and 20.3-33.5% with nodal involvement in breast cancer for

receptor positivity64,67.It has been observed that cases with more than 4 nodes involved, did

not express hormone receptors as compared to those with less than 4 nodes involved 57.

Molecules that have been extensively studied as immunohistochemical markers, but presently

have not found widespread clinical use in breast cancers include MUC 1 (a glycosylated

mucin protein), peptide growth factors and their receptor (eg. EGF, EGFR, TGF-α, TGF-β),

other oncogenes (eg. ras, c-myc), p53 tumour suppressor gene, cell proliferation markers (eg.

Ki67, PCNA) and other molecules (eg. metalloproteases, intermediate filament proteins,

basement membrane components, CEA, cathepsin D) 68

OTHER CLINICOPATHOLOGICAL PROGNOSTIC PARAMETERS:

Patient's age:

Women who are younger than 50 years of age at the time of diagnosis have the best

prognosis. Relative survival declines after the age of 50 years and is particularly low in older

women.69 As far as very young women (<=35 years of age) are concerned, some studies have

shown a prognosis similar to that in older patients,70 whereas others have shown a

significantly higher risk for recurrence and distant metastases, related to the fact that these

patients tend to have higher grade tumors.

Pregnancy and oral contraceptives.

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Carcinoma of the breast manifesting during pregnancy or lactation is generally an aggressive

tumor with low expression of hormone receptors and high expression of Her2neu,71 and that it

is associated with an overall poorer prognosis. No convincing evidence has been found that

prior use of oral contraceptive agents has an effect on the evolution or survival of breast

carcinoma.72

Genetic Factors:

A genetic basis for differences in rates of cancer progression is suggested by analysis of

LOH in primary and locally recurrent lesions. Regitnig et al. 73 studied primary and recurrent

tumor specimens from 26 patients and reported that all LOH identified in the primary tumor

was also present in the local recurrence but that there was a significant increase in “total

LOH” in recurrent tumors. Early recurrence was associated with LOH at specific loci (TP53

and D5S107). Lymph node metastases were associated with LOH at these sites and also at

D35.

The impact on prognosis of mutations in specific genes such as BRCA1 and BRCA2 is the

subject of active investigation. BRCA-associated breast carcinomas are significantly more

likely than non-BRCA-associated carcinomas to have poorly differentiated histologic grade,

lack estrogen and progesterone receptors, be HER2/neu negative, and manifest high Ki67-

labeling indicative of a high proliferative rate74,75. The differences are greatest in BRCA1-

associated carcinomas, which have higher frequencies of medullary carcinoma, poorly

differentiated histologic grade, high mitotic rate, prominent lymphocytic infiltrates, and

necrosis than BRCA2-associated carcinomas.

Bilaterality

The impact of bilateral breast carcinoma on prognosis has received considerable attention.

Among women treated by mastectomy, prognosis is similar after unilateral and bilateral

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disease, compared on the basis of the higher stage tumor in bilateral cases 76. When compared

to patients with unilateral carcinoma, patients with bilateral carcinoma had a greater

frequency of multicentricity in one or both breasts (19% vs. 3%, respectively)77.

Method of Tumor Detection and Screening

The method of tumor detection influences prognosis and disease-free survival. Among

patients diagnosed before widespread mammography screening, tumor detection by clinical

breast examination was associated with a significant reduction in recurrence compared with

detection by self-palpation78. Screening examinations employing mammography with or

without physical examination have been shown to reduce mortality due to breast carcinoma

in the screened population.

When compared to breast carcinomas presenting clinically, carcinomas detected by screening

with mammography tend to be smaller, to be lower grade, and to have fewer nodal

metastases. Hence, the advantage conferred by screening was dependent on detecting smaller

tumors.

Time to Recurrence

The interval to recurrence (disease-free survival or recurrence-free interval) and length of

survival are basic measurements of prognosis. In general, these are closely related; hence

factors associated with a high frequency of recurrence correlate with reduced survival and

overall mortality due to the disease.

Local Radiotherapy

Radiotherapy administered to the chest wall after mastectomy or to the breast after

lumpectomy reduces the risk of local recurrence at these sites. Whether this beneficial effect

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on local recurrence is translated into improved overall survival in patients who receive

radiation to the chest wall after mastectomy remains controversial, and the benefit, if any,

may not be sufficient to offset potential complications such as cardiovascular disease79.

Local Recurrence in Conserved Breast or Chest Wall

In patients treated by breast-conserving surgery and radiation therapy, the time to breast

recurrence is significantly related to the risk of systemic metastases and survival at 5 years 80.

Patients with breast recurrences 2 to 4 years after diagnosis have a significantly greater risk

of developing systemic metastases and poorer survival than those who manifest breast

recurrence more than 4 years after initial treatment.

Impact of Therapy on Prognosis

It is also important to consider the impact of primary treatment on the evaluation of

prognostic factors. Radical mastectomy was, until the 1960s, the most widely employed form

of primary treatment. There has been major changes in treatment, with a shift from total

mastectomy to partial mastectomy, quadrantectomy, or lumpectomy combined with primary

radiation therapy and axillary dissection or sentinel lymph node (SLN) mapping. The

evaluation of prognostic factors has been further complicated by the introduction of adjuvant

hormonal and chemotherapy for women with nodal metastases and for many patients with

uninvolved lymph nodes. The extent to which the prognostic importance of conventional

pathologic parameters influences response to adjuvant chemotherapy is uncertain.

Gross Pathology

Invasive duct carcinoma typically forms a solid tumor. Carcinomas with a relatively abundant

scirrhous or fibrotic stroma can be extremely firm to hard, with a gray to white surface. When

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there is prominent elastosis of the stroma, a yellow tinge may be observed. Chalky white

streaks in the tumor tissue are usually indicative of necrosis, calcification, or elastosis.

Carcinomas with less abundant stroma that are composed largely of neoplastic cells and

inflammatory cells tend to be softer and tan.

Tumor Size

The measured gross size represented by the largest diameter of a mammary carcinoma is one

of the most significant prognostic variables. Numerous studies have shown that survival

decreases with increasing tumor size and that there is a coincidental rise in the frequency of

axillary nodal metastases81,82,84. This phenomenon applies not only to the overall spectrum of

primary tumor size, but also within subsets such as those defined by TNM (tumor-node-

metastasis) staging. For example, among T 1 breast carcinomas (2 cm or less in diameter),

there is a significant relationship between size, the frequency of nodal metastases, and

prognosis when the tumors are stratified in 5-mm groups82.

Tumor Configuration or Shape

Some investigators have observed a more favorable prognosis associated with circumscribed

carcinomas determined by gross inspection of the tumor or by mammography. Infiltrative

tumors tend to be larger when detected, and they are more likely to have axillary lymph node

metastases than those with circumscribed margins83. Tumors with a stellate configuration in

which there is focal necrosis were found to have an especially poor prognosis.

Fitzgibbons et al considered prognostic and predictive factors in breast cancer and stratified

them into categories. Category 1 (prognostically important and useful in management)

included tumour size, nodal status, histological grade, histological grade, mitotic figure count

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and hormone receptor status. Category 2 (extensively studied but not that important) included

Her 2 neu, p53, lymphatic or vascular channel invasion and category 3 (not sufficiently

studied) included DNA ploidy analysis, angiogenesis, EGFR, TGF alpha etc.84

As seen from the literature review , most studies have concentrated on the tumour

characterstics, grade and individual tumour cell histological, immunohistochemical and

genetic characterstics, only few studies have focussed on the characteristics of tumour

margins of breast carcinoma.The present study focusses on tumour margin type, tumour

budding, adipocytic infiltration and peritumoural lymphocytic infiltrate.

TUMOUR BUDDING

Epithelial mesenchymal transition (EMT) is a biological process allowing a polarized cell,

normally interacting with a basement membrane, to assume a mesenchymal phenotype

characterized by increased migratory capacity, invasiveness, increased resistance to apoptosis

and increased production of extracellular matrix (ECM) components 85. The completion of

EMT is signaled by the degradation of the basement membrane and formation of a

mesenchymal cell. Highly relevant for embryogenesis and wound healing, EMT has also

been proposed as a critical mechanism for the acquisition of malignant phenotypes by

epithelial cells86. EMT-derived tumor cells occurring at the invasive tumor front are thought

to be those cells entering into subsequent steps of invasion and metastasis. Moreover, these

cells have been shown to establish secondary colonies at distant sites that histopathologically

resemble the primary tumor of origin through a process known as mesenchymal epithelial

transition (MET).

In colorectal cancer, EMT-derived tumor cells are represented histopathologically by the

presence of tumor buds and are reported to occur in 20-40% of tumours. Occurring

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predominantly at the invasive front, the identification of tumor buds, defined as single cells or

clusters of up to 5 cells, can be done using standard H&E-stained slides or facilitated by

using pan-cytokeratin stains. In addition, these budding cells can often be seen in the

company of “pseudo-like” cytoplasmic protrusions in direct contact with adjacent structures

which are thought to be a marker of an activated budding phenotype associated with cell

motility and increased invasiveness 87. The study of EMT and its related signaling pathways

could provide the first clues regarding the molecular and genetics events promoting tumor

budding.

Tumor budding at the invasive front has been recognized as an adverse parameter and an
88
“additional” prognostic factor by the International Union against Cancer (UICC) . High-

grade tumor budding, irrespective of the definition, has been consistently linked to lymph

node metastasis , distant metastasis, local recurrence and correlates with the distance of ,

tumor invasion beyond the outer border of the muscularis propria in colorectal carcinoma.

The prognostic and independent effect of tumor budding on outcome has been investigated

by several study groups.89

Tumor budding introduced as a reliable histopathological hallmark to estimate the

aggressiveness of rectal cancer, was initially shown to have a superior prognostic value when

compared to other histopathological characteristics, including tumor differentiation and

venous invasion. Yusra, Shuho samba and Hiroshi Yokozaki in their study concluded that at

the invasive front of colorectal carcinoma, the existence of tumor budding, the detachment

and migration of small clusters of tumor cells from the neoplastic epithelium, correlates with

high incidence of local invasion and distant metastasis.90

Prall F in his study discussed the morphological features of tumour budding. He strongly

advocated tumour budding as a promising factor beyond TNM in colorectal cancer.37

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Fabio Grizzi, Giuseppe Celesti, Gianluca Basso, Luigi Laghi in their study considered tumor

budding as an independent adverse prognostic factor in Colorectal cancer that may allow for

stratification of patients into and also potentially guide treatment decisions, especially in T2-

T3 N0 (stage Ⅱ) CRCs.92

To date, the prognostic significance of tumor budding has mainly been examined in the field

of colorectal cancer. Recently, the extent of budding was also identified as an independent

predictive factor for node metastases in early colorectal cancer. Due to its significant

prognostic value, tumor budding had been recommended to be included in the reporting of

colorectal cancer93.

In recent study of 2013 Fenli Liang, Wei Caoc, Yili Wang et al94 investigated the prognostic

value of tumor budding in IDC-NOS. Three main findings were presented: First, the grade of

tumor budding in IDC-NOS was a reproducible histopatholgical index and was associated

with the aggressive behaviors and poor prognosis. Second, the budded cells in IDC NOS

showed reduced membrane E-cadherin expression, increased cytoplasmic vimentin

expression and decreased nuclear Ki67 expression as compared with tumor cells in center

areas. Third, the grade of budding was confirmed as a significant prognostic factor

independent of classical variables, such as tumor size, node status, and LVI status. They

concluded that tumor budding was a valuable prognostic factor in breast cancer.

Previous reports have suggested budded cells in breast cancer displayed the EMT (Epithelial

mesenchymal transition) like molecular phenotype. Since EMT endows cells with migratory

and invasive properties, tumor budding at the invasive margin was postulated as the first step

of invasion and metastasis95, which implied that tumor budding would be a more sensitive

indicator of aggressiveness as compared with traditional histopathological variables, such as

node metastases and LVI. In addition, they validated that budded cells in breast cancer

displayed lower proliferation activity than tumor cells in the center areas, which is consistent

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with previous reports on colorectal cancers. This suggested that invasion and proliferation

might be two contrasting events in tumor progression. As tumor budding is a potential

oncotarget, more detailed molecular properties of budded tumor cells deserve to be elucidated

in the future95.

According to the guidelines of a prognostic factors study, a potential prognostic factor should

first be reproducible and widely available with quality control 96. To date, consensus on the

definition and quantification of tumor budding has not been reached. Besides the counting

method proposed by Uneo et al.97, other criteria, such as the scoring method and the rapid bud

count method etc were also used. However, despite the lack of standardization, nearly all the

reported evaluating systems presented with a good reproducibility, and almost all the studies

presented with a significant association between tumor budding and the adverse clinical

outcome. In the study done by Fenli Lianga, Wei Caoc, Yili Wang et al94 , they adopted the

counting method, and selected the maximal value of five densest budding fields as the

number of tumor budding. This method is easy to be implemented and showed a good inter

observer reproducibility. The findings in the study disclosed the clinical significance of the

minor components in carcinoma. Although grading and typing in breast cancer constitute the

major content of a pathological report, the minor but key poorly differentiated elements may

determine the clinical outcome. The budded tumor cells at the margin had been demonstrated

as the poorly differentiated component, which could illustrate the aggressive behavior and

adverse prognosis in cancers. This new histological parameter could be used to complement

the traditional histopathological prognostic factors in breast carcinoma.

Recent study done by Yan-gao Man attempted to elucidate the early alterations of the

myoepithelial cells and their impact on associated epithelial cells during tumor progression.

Although tumor cell budding from focally disrupted tumor capsules was seen in all breast

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cancer subtypes, the frequency and size of the capsule disruptions and the cell projections

varied significantly among different subtypes. Large focal disruptions and large budding cell

projections (with about 100 individual cells) were exclusively or preferentially seen in duct-

derived and clinically more aggressive subtypes. Clinically indolent tubular carcinoma had

the lowest frequency of tumor capsule disruptions with budding tumor cells.98

ADIPOSE TISSUE INVASION

The breast stroma histologically consists of fibrous tissues and adipocytes in variable

proportions, and the adipocytes physiologically increase with aging in older women.

Generally, when intraductal carcinoma cells infiltrate the breast stroma, the cells initially

penetrate the fibrous tissues, followed by the fibroadipose tissues, and, finally, the adipose

tissues in breast cancer.

The International Union Against Cancer classification has shown the significance of chest

wall and skin invasions in evaluating local tumor extension in breast cancer. Some

investigators noted that pathologic evidence of fat invasion, such as scattered invasion into fat

tissues99 and the invasive length of fat invasion, was related to a poor prognosis. However,

the prognostic significance of adipose tissue invasion (ATI) at the tumor margin has not been

fully evaluated in breast cancer, and the biologic characteristics of tumors with ATI are also

insufficiently known.

Meanwhile, many studies have been conducted to define the significance of lymphovascular

invasion. Peritumoral lymphatic vessel invasion (LVI) is associated with a higher frequency

of nodal metastasis and has also been cited as a risk factor in breast carcinoma.100

Junzo Yamaguchi, Hiroshi Othani, Kazukuni Nakamura10 revealed adipose tissue invasion

(ATI) of cancer cells at the tumour margin was independently associated with nodal

involvement in patients with invasive ductal carcinoma of the breast. Second, patient age

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and invasive tumour size were significant factors for ATI. Finally, patients without ATI had

an excellent prognosis. Marginal ATI may lead to a larger contact area between cancer cells

and the peritumoral functional lymphatic endothelium.10

The presence of ATI may reflect the infiltrating growth pattern of cancer cells at the marginal

site, while the absence of ATI may be associated with an expanding growth pattern or the

existence of abundant fibrous tissues surrounding the ducts and lobules as a result of other

underlying conditions such as fibrocystic changes and fibrous mastopathy. Furthermore,

stromal reaction patterns, including edema and desmoplasia, in invasive cancer may affect

ATI or prognosis in patients with breast cancer.

In most patients with breast cancer, only fibrous or fibro-adipose tissue invasion by cancer

cells appears insufficient for nodal metastasis. The true mechanisms of ATI-associated nodal

metastasis are unknown, but distinct molecular mechanisms are likely to be concerned with

ATI- and peritumoral LVI-associated nodal metastasis. Aromatase activity and

adipocytokines such as leptin in mammary adipose tissues may participate in ATI-associated

nodal involvement. Several investigators have reported that aromatase activity and its

expression in breast adipose tissues are most prominent in regions proximal to the tumor in

breast cancer.102

There is a possibility that tissue estrogen concentration is higher in breast carcinoma with

ATI. This may cause the aggressive biologic behavior of breast cancer with ATI and the

subsequent involvement of the lymphatic network.103 In addition, mammary adipose tissue is

an important source of various adipocytokines, including leptin. Leptin is one of the

neurohormone regulators in the hypothalamus and is necessary to normal mammary gland

development and lactation. It might be also involved in carcinogenesis and progression in

breast cancers. Such paracrine adipocytokines may contribute to tumor aggressiveness and

ATI-associated nodal metastasis.

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104
Alkarain et al recently reported that the lymphatic endothelial marker D2-40 is useful for

detecting intratumoral obliterated LVI that cannot be visualized on H&E-stained sections.

These intratumoral LVIs in adipose tissue may be associated with nodal metastasis. Taken

together, further investigations on the molecular mechanisms of vessel invasion and genetic

participation in the invasive process are essential for anticancer therapy, including new

molecular target therapy

The precise evaluation of marginal ATI and peritumoral LVI will prove to be useful in the

formulation of therapeutic strategies and the prediction of which patients with breast cancer

have an excellent prognosis.

Enbo Liu, Fahumiya Samad, and Barbara M Mueller suggest that local interactions between

adipocytes and tumor cells are sufficient to promote the growth of hormone-dependent breast

cancer. They also demonstrated that leptin signaling in adipocytes induces aromatase

expression, expected to result in higher estrogen in the microenvironment thus enabling

mammary tumorigenesis.105

TUMOUR BORDER

Since the discovery that infiltrative margins indicate a poor prognosis in colorectal

carcinoma, tumor margin histology has been interpreted to indicate tumor aggressiveness or

invasiveness. Till recently, the prognostic significance of this margin type has been approved

from several studies in various malignancies106. Including this one, all studies have classified

the margin types into two categories, (expanding vs infiltrative type) despite of histological

variation of primary tumor; and the criteria for margin classification were also not much

different.

The majority of invasive duct carcinomas can be described on the basis of gross tumor

configuration as stellate (spiculated, infiltrative, radial, serrated), circumscribed (rounded,

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pushing, encapsulated, smooth), or having a mixed contour. Approximately one-third of the

tumors have grossly circumscribed margins. A minority of tumors have indistinct borders and

cannot be described in these terms. In general, the gross appearance of the tumor duplicates

the configuration visualized by mammography. However, carcinomas that appear to have

circumscribed margins grossly or mammographically may exhibit an invasive growth pattern

when studied microscopically. Some investigators have observed a more favorable prognosis

associated with circumscribed carcinomas determined by gross inspection of the tumor or by

mammography. Infiltrative tumors tend to be larger when detected, and they are more likely

to have axillary lymph node metastases than those with circumscribed margins 84. Tumors

with a stellate configuration in which there is focal necrosis were found to have an especially

poor prognosis.

Carter D et al in their study reviewed the pathological findings of 330 patients who

underwent radical mastectomy and followed them for 10 years. They studied presence or

absence of tumour necrosis and character of tumour border in all these cases. They concluded

that the tumour with necrosis and an infiltrating border showed significantly more aggressive

behaviour than did those tumours with a rounded border and no necrosis.83

PERITUMOURAL LYMPHOCYTIC INFILTRATE.

Inflammatory cells have gained a renewed interest in breast cancer research due to increasing

understanding of their role in tumor development, and also due to increased ability to

identify each cell type. Leukocyte infiltrate includes a variable representation of leukocytes,

including macrophages, neutrophils, mast cells, and T and B-lymphocytes 107. There are

evidences indicating that different types of breast carcinomas may have different types of

leukocyte infiltrate with distinct abilities to control tumor growth according to their tumor

dissemination. Thus, whereas macrophages are known to have several pro-tumor functions

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and macrophage infiltration has also been associated with worse prognosis, it has been

reported that both T and B-lymphocytes perform an important immunological function by

inhibiting cancer development and progression108.

Inflammation is now considered a hallmark of cancer and can play a role in all aspects of

tumor biology, including initiation, promotion, angiogenesis, and metastasis 109. It is known

that the activation of oncogenes can trigger the production of inflammatory molecules and the

recruitment of inflammatory cells. But the potential effects of the inflammatory cell infiltrate

in breast cancer seem to be diverse and complex. This is of special interest because the

invasive front is the area where some of the most important interactions between cancer cells

and the tumor supporting stroma take place.

There are data indicating that, depending on the cell type present and their functional profile,

inflammatory cells can either suppress or promote tumor growth. It has been reported that

activated B cells can mediate tumor regression by itself and confers host T cell antitumor

immunity. Tumor-associated macrophages produce a variety of cytokines and chemokines, as

well as growth factors for both epithelial and endothelial cells, which play a key role in tumor

growth and metastasis

Bin jiang et al in their study on tumour infiltrating immune cells concluded that aberrant

immune cell infiltration may have the same destructive impact on the capsule of all epithelial

derived tumours. This may favour the proliferation of tumour stem or progenitor cells

overlying these focal disruptions. These proliferating epithelial tumour cells subsequently

disseminate from the focal disruption leading to tumour invasion and metastasis. Their study

also revealed that infiltrating immune cells were preferentially associated with epithelial

capsules that show distinct degenerative alterations and infiltrating immune cells appeared to

facilitate tumor stem cell proliferation, budding, and dissemination.110

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Shuzhen Liu et al in their study of 3403 cases found CD8+ tumor infiltrating lymphocytes

were identified in an intratumoral pattern in 32% and stromal pattern in 61% of the cases. In

the whole cohort, the presence of tumor-infiltrating lymphocytes was significantly correlated

with young age, high grade, estrogen receptor negativity, human epidermal growth factor

receptor-2 positivity and core basal intrinsic subtype, and was associated with superior breast

cancer specific survival.111

In their study, Sahar M.A. Mahmoud et al showed that tumor-infiltrating CD8 lymphocytic

density is significantly associated with improved clinical outcome in a large cohort of

patients with breast cancer, independent of the standard prognostic and predictive factors:

tumor grade, lymph node stage, size, vascular invasion, and HER 2 neu status. Their results

suggest that the cell-mediated immune reaction has an important role in breast cancer.7

Macchetti AH et al used dual-flow cytometry for accurate detection of selected lymphocyte

subsets, especially the subpopulations of CD3+ tumor infiltrating lymphocytes after enzyme

digestion method. In their study, the immunophenotyping of cell types revealed a familiar

subdivision of lymphocytes. The tumor infiltrating lymphocytes, like peripheral blood

lymphocytes, consisted mainly of T cells expressing the CD3 antigen, and there was a

relative paucity of tumor-infiltrating B lymphocytes and NK cells. Their analysis revealed an

association of higher levels of tumor infiltrating CD4+ T lymphocytes with lymph node

involvement by metastasis8.

Rafal Matkowsi et al in their study concluded that in early breast cancer, the presence of

CD8+ and CD4+ cells correlates with lymph node involvement and unfavourable prognosis,

suggesting that immune response has an influence on the behaviour of malignant breast

tumors. The analysis of tumor-infiltrating immune cells may predict lymph node involvement

and higher risk of recurrence.9

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Given the heterogenous nature of carcinoma breast in terms of response to treatment, efforts

have been made to identify histological features to predict the aggressiveness and select

patients for aggressive therapy. This study is designed to evaluate the characteristics of

invasive front which is the site of tumour host interaction to be able to add easily identifiable

additional prognostic factors to improve the clinical management of the patients with this

disease.

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